The beta2-adrenoceptor (?2-AR) is a G protein-coupled receptor (GPCR) that mediates physiological responses to noradrenaline in the brain and critically modulates long-term potentiation, synaptic plasticity and memory acquisition. Disturbances in this pathway have been implicated in mental illnesses such as Alzheimer's disease, Parkinson's disease, Down's syndrome, and post-traumatic stress disorder. Therefore, elucidating the molecular mechanisms that control ?2-AR signaling and mediate its functional consequences in neurons will illuminate how cells execute critical brain functions and will enhance our ability to develop more specific and efficient therapies for memory disorders and mental illnesses. It was previously thought that ?2- AR signaling occurred via cyclic AMP (cAMP) production strictly at the plasma membrane, but this canonical view has recently been challenged by evidence that ligand-dependent stimulation of ?2-ARs also occurs on endosomes following agonist-induced endocytosis. I have demonstrated in HEK293 cells that endosomal ?2- AR/cAMP signaling is selectively linked to the receptor-dependent transcriptional response, whereas the plasma membrane signal is effectively uncoupled from this response. My immediate goal is to investigate the functional consequences of compartmentalized ?2-AR/cAMP signaling in primary neurons. My long-term goal is to understand how the spatiotemporal dynamics of ?2-AR signaling contribute to higher-level neuronal functions. This K99/R00 award will allow me to achieve the following career goals: 1) gain training in neuroscience, 2) obtain additional training in high-throughput genetic analysis using CRISPR interference, 3) develop grant-writing, mentoring, and communication skills I will need in order to become a successful independent researcher. During the mentored phase of the award, I will determine if the transcriptional response to ?2-AR activation in primary hippocampal neurons is governed by spatial encoding of cAMP signals (Aim 1). Next, I will systematically define the phosphoproteomic response to ?2-AR signaling from distinct cellular compartments, and test the hypothesis that signaling from the plasma membrane (which does not efficiently activate transcription) preferentially stimulates phosphorylation of distinct neuronal target proteins (Aim 2). During the independent phase of the award, I will identify genes that regulate the spatial specificity of ?2-AR signaling by conducting a large-scale CRISPR-based genetic analysis (Aim 3). The biochemical and systems-level analysis of ?2-AR signaling accomplished with this award will help illuminate how the spatial regulation and molecular consequences of this pathway contribute to critical neuronal functions at the cellular level. Future studies based on these results will elucidate the precise mechanisms by which spatially biased signal transduction is achieved and investigate how the compartment-specific ?2-AR/cAMP targets and the mechanisms that regulate them ultimately contribute to higher-level neuronal functions.